本文選題：血管內(nèi)皮生長因子 + 克隆　； 參考：《大連醫(yī)科大學(xué)》2005年碩士論文

【摘要】：目的:構(gòu)建人血管內(nèi)皮生長因子 165(Vascular endothelial growthfactor 165,VEGF165)原核表達(dá)載體并在大腸桿菌中誘導(dǎo)表達(dá)。構(gòu)建VEGF165 真核表達(dá)載體并將其轉(zhuǎn)入人慢性髓系白血病急變細(xì)胞株K562 中,研究其對 K562 增殖和凋亡的影響,進(jìn)一步驗(yàn)證 VEGF165通過自分泌途徑對腫瘤生長發(fā)揮作用,從而為臨床上惡性血液病抗血管新生治療的部分機(jī)理提供實(shí)驗(yàn)依據(jù)。 方法:用 RT-PCR 法從人白血病細(xì)胞株 TF1 擴(kuò)增 VEGF165 完整編碼序列(不包含前面 81bp 的信號肽序列及終止密碼子),將其插入質(zhì)粒pUCmT中并測序鑒定。將測序正確的pUCmT-VEGF165與PET20b(+)均用 EcoRI 和 SalI 雙酶切,回收純化目的基因片段與表達(dá)載體,構(gòu)建VEGF165 的 原 核 表 達(dá) 載 體 PET20b-VEGF165, 轉(zhuǎn) 化 大 腸 桿 菌BL21(DE3)pLysS,IPTG 誘導(dǎo)表達(dá),表達(dá)產(chǎn)物用 Ni-NTA Resin 純化,SDS-PAGE 及 Western Blot 鑒定重組蛋白。用 RT-PCR 法從人白血病細(xì)胞株 HL60 擴(kuò)增 VEGF165 完整編碼序列, 將其插入質(zhì)粒 pUCmT 中并測序鑒定。將測序正確的 pUCmT-VEGF165 與 pcDNA3.1(-)均用 EcoR I和 HindⅢ雙酶切,回收純化目的基因片段與表達(dá)載體,構(gòu)建 VEGF165的真核表達(dá)載體 pcDNA3.1-VEGF165。用脂質(zhì)體介導(dǎo)的方法轉(zhuǎn)染人慢性髓系白血病急變細(xì)胞株 K562,G418 篩選轉(zhuǎn)染陽性的細(xì)胞株。RT-PCR和Western Blot檢測VEGF165在RNA水平和蛋白水平的表達(dá)。用MTT
[Abstract]:Objective: to construct the prokaryotic expression vector of human vascular endothelial growth factor 165 (Vascular endothelial growthfactor 165, VEGF165) and to induce expression in Escherichia coli. The VEGF165 eukaryotic expression vector was constructed and transferred into the human chronic myeloid leukemia acute cell strain K562 to study the effect of the eukaryotic expression vector on the proliferation and apoptosis of K562 and further verify VE. GF165 can play a role in tumor growth through autocrine pathway, thus providing experimental evidence for the clinical mechanism of anti angiogenesis therapy for hematological malignancies.
Methods: RT-PCR method was used to amplify the complete coding sequence of VEGF165 from human leukemia cell line TF1 (not including the sequence of signal peptide and terminating codon of the preceding 81bp) and insert it into plasmid pUCmT and sequenced and identified. The correct pUCmT-VEGF165 and PET20b (+) were sequenced with EcoRI and SalI double enzyme, and the purified target gene fragment and expression were recovered. Vector, the prokaryotic expression vector PET20b-VEGF165 of VEGF165 was constructed, the expression of Escherichia coli BL21 (DE3) pLysS, IPTG was induced, the expression products were purified with Ni-NTA Resin, SDS-PAGE and Western Blot were used to identify the recombinant protein. The whole coding sequence was amplified by RT-PCR method and inserted into the plasmid. PUCmT and sequencing identification. The correct pUCmT-VEGF165 and pcDNA3.1 (-) were sequenced with EcoR I and Hind III double enzyme, the purified target gene fragment and expression vector were purified, and the eukaryotic expression vector of VEGF165 was constructed by liposome mediated transfection of K562 in human chronic myeloid leukemia rapid cell line, and G418 screening turned. .RT-PCR and Western Blot were used to detect the expression of VEGF165 at RNA level and protein level. MTT was used.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R341

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 傅建新,王瑋,白霞,王玲,朱子玲,陳子興,阮長耿;腫瘤細(xì)胞系血管內(nèi)皮生長因子及其受體共表達(dá)的研究[J];癌癥;2002年11期

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本文編號：1966739